Inhibition of angiogenesis in disease states with an anti-αvβ3 monoclonal antibody

ABSTRACT

The present invention describes methods for inhibition angiogenesis in tissues using vitronectin α v β 3  antagonists, and particularly for inhibiting angiogenesis in inflamed tissues and in tumor tissues and metastases using therapeutic compositions containing α v β 3  antagonists.

This application is a continuation of U.S. application Ser. No.08/210,715, filed Mar. 18, 1994, now issued as U.S. Pat. No. 5,753,230.

This invention was made with government support under Contract No. CA45726 by the National Institutes of Health. The government has certainrights in the invention.

TECHNICAL FIELD

The present invention relates generally to the field of medicine, andrelates specifically to methods and compositions for inhibitingangiogenesis of tissues using antagonists of the vitronectin receptorα_(v)β₃.

BACKGROUND

Integrins are a class of cellular receptors known to bind extracellularmatrix proteins, and therefore mediate cell-cell and cell-extracellularmatrix interactions, referred generally to cell adhesion events.However, although many integrins and the ligands that bind an integrinare described in the literature, the biological function of many of theintegrins remains elusive. The integrin receptors constitute a family ofproteins with shared structural characteristics of noncovalentheterodimeric glycoprotein complexes formed of α and β subunits.

The vitronectin receptor, named for its original characteristic ofpreferential binding to vitronectin, is now known to refer to threedifferent integrins, designated α_(v)β₁ α_(v)β₃ and α_(v)β₅. Horton,Int. J. Exp. Pathol., 71:741-759 (1990). α_(v)β₁ binds fibronectin andvitronectin. α_(v)β₃ binds a large variety of ligands, including fibrin,fibrinogen, laminin, thrombospondin, vitronectin, von Willebrand'sfactor, osteopontin and bone sialoprotein I. α_(v)β₅ binds vitronectin.The specific cell adhesion roles these three integrins play in the manycellular interaction in tissues is still under investigation, but it isclear that there are different integrins with different biologicalfunctions.

One important recognition site in the ligand for many integrins is thearginine-glycine-aspartic acid (RGD) tripeptide sequence. RGD is foundin all of the ligands identified above for the vitronectin receptorintegrins. This RGD recognition site can be mimicked by polypeptides(“peptides”) that contain the RGD sequence, and such RGD peptides areknown inhibitors of integrin function. It is important to note, however,that depending upon the sequence and structure of the RGD peptide, thespecificity of the inhibition can be altered to target specificintegrins.

For discussions of the RGD recognition site, see Pierschbacher et al.,Nature, 309:30-33 (1.984), and Pierschbacher et al., Proc. Natl. Acad.Sci. USA, 81:5985-5988 (1984). Various RGD polypeptides of varyingintegrin specificity have also been described by Grant et al., Cell,58:933-943 (1989), Ruggeri et al., and Aumailley et al., FEBS Letts.,291:50-54 (1991), and in U.S. Pat. Nos. 4,517,686, 4,578,079, 4,589,881,4,614,517, 4,661,111, 4,792,525, 4,683,291, 4,879,237, 4,988,621,5,041,380 and 5,061,693.

Angiogenesis is a process of tissue vascularization that involves thegrowth of new developing blood vessels into a tissue, and is alsoreferred to as neo-vascularization. The process is mediated by theinfiltration of endothelial cells and smooth muscle cells. The processis believed to proceed in any one of three ways: The vessels can sproutfrom pre-existing vessels, de-novo development of vessels can arise fromprecursor cells (vasculogenesis), or existing small vessels can enlargein diameter. Blood et al., Bioch. Biophys. Acta, 1032:89-118 (1990).Vascular endothelial cells are known to contain at least fiveRGD-dependent integrins, including the vitronectin receptor (α_(v)β₃ orα_(v)β₅ ), the collagen Types I and IV receptor (α₁β₁), the lamininreceptor (α₂β₁), the fibronectin/laminin/collagen receptor (α₃β₁) andthe fibronectin receptor (α₅β₁). Davis et al., J. Cell. Biochem.,51:206-218 (1993). The smooth muscle cell is known to contain at leastsix RGD-dependent integrins, including α₅β₁, α_(v)β₃ and α_(v)β₅.

Angiogenesis is an important process in neonatal growth, but is alsoimportant in wound healing and in the pathogenesis of a large variety ofclinical diseases including tissue inflammation, arthritis, tumorgrowth, diabetic retinopathy, macular degeneration by neovascularizationof retina and the like conditions. These clinical manifestationsassociated with angiogenesis are referred to an angiogenic diseases.Folkman et al., Science, 235:442-447 (1987). Angiogenesis is generallyabsent in adult or mature tissues, although it does occur in woundhealing and in the corpeus leuteum growth cycle. See, for example, Moseset al., Science, 248:1408-1410 (1990).

It has been proposed that inhibition of angiogenesis would be a usefultherapy for restricting tumor growth. Inhibition of angiogenesis hasbeen proposed by (1) inhibition of release of “angiogenic molecules”such as βFGF, (2) neutralization of angiogenic molecules, such as by useof anti-βFGF antibodies, and (3) inhibition of endothelial cell responseto angiogenic stimuli. This latter strategy has received attention, andFolkman et al., Cancer Biology, 3:89-96 (1992), have described severalendothelial cell response inhibitors, including collagenase inhibitor,basement membrane turnover inhibitors, angiostatic steroids,fungal-derived angiogenesis inhibitors, platelet factor 4,thrombospondin, arthritis drugs such as D-penicillamine and goldthiomalate, vitamin D₃ analogs, alpha-interpheron, and the like thatmight be used to inhibit angiogenesis. For additional proposedinhibitors of angiogenesis, see Blood et al., Bioch. Biophys. Acta.,1032:89-118 (1990), Moses et al., Science, 248:1408-1410 (1990), Ingberet al., Lab. Invest., 59:44-51 (1988), and U.S. Pat. Nos. 5,092,885,5,112,946, 5,192,744, and 5,202,352. None of the inhibitors ofangiogenesis described in the foregoing references are targeted atinhibition of α_(v)β₃.

RGD-containing peptides that inhibit vitronectin receptor α_(v)β₃ havealso been described. Aumailley et al., FEBS Letts., 291:50-54 (1991),Choi et al., J. Vasc. Surg., 19:125-134 (1994), and Smith et al., J.Biol. Chem., 265:12267-12271 (1990). However, the role of the integrinα_(v)β₃ in angiogenesis has never been suggested nor identified untilthe present invention.

Inhibition of cell adhesion in vitro using monoclonal antibodiesimmunospecific for various integrin α or β subunits have implicatedα_(v)β₃ i in cell adhesion of a variety of cell types includingmicrovascular endothelial cells. Davis et al., J. Cell. Biol.,51:206-218 (1993). In addition, Nicosia et al., Am. J. Pathol.,138:829-833 (1991), described the use of the RGD peptide GRGDS (SEQ IDNO 15) to in vitro inhibit the formulation of “microvessels” from rataorta cultured in collagen gel. However, the inhibition of formulationof “microvessels” in vitro in collagen gel cultures is not a model forinhibition of angiogenesis in a tissue because it is not shown that themicrovessel structures are the same as capillary sprouts or that theformulation of the microvessel in collagen gel culture is the same asneo-vascular growth into an intact tissue, such as arthritic tissue,tumor tissue or disease tissue where inhibition of angiogenesis isdesirable.

Therefore, other than the studies reported here, Applicants are unawareof any other demonstration that angiogenesis could be inhibited in atissue using inhibitors of cell adhesion. In particular, it has neverbeen previously demonstrated that α_(v)β₃ function is required forangiogenesis in a tissue or that α_(v)β₃ antagonists can inhibitangiogenesis in a tissue.

BRIEF DESCRIPTION OF THE INVENTION

The present invention disclosure demonstrates that angiogenesis intissues requires integrin α_(v)β₃, and that inhibitors of α_(v)β₃ caninhibit angiogenesis. The disclosure also demonstrates that antagonistsof other integrins, such as α_(v)β₅ or α_(v)β₁, do not inhibitangiogenesis, presumably because these other integrins are not essentialfor angiogenesis to occur.

The invention therefore describes methods for inhibiting angiogenesis ina tissue comprising administering to the tissue a composition comprisingan angiogenesis-inhibiting amount of an α_(v)β₃ antagonist.

The tissue to be treated can be any tissue in which inhibition ofangiogenesis is desirable, such as diseased tissue whereneo-vascularization is occurring. Exemplary tissues include inflamedtissue, solid tumors, metastases, and the like tissues.

An α_(v)β₃ antagonist for use in the present methods is capable ofbinding to α_(v)β₃ and competitively inhibiting the ability of α_(v)β₃to bind to a natural ligand. Preferably, the antagonist exhibitsspecificity for α_(v)β₃ over other integrins. In a particularlypreferred embodiment, the α_(v)β₃ antagonist inhibits binding offibrinogen or other RGD-containing ligands to α_(v)β₃ but does notsubstantially inhibit binding-of fibronectin to α_(IIb)β₃. A preferredα_(v)β₃ antagonist can be a polypeptide or a monoclonal antibody, orfunctional fragment thereof, that immunoreacts with α_(v)β₃.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming a portion of this disclosure:

FIGS. 1A-1D illustrate the tissue distribution of the integrin subunits,β₃ and β₁, in normal skin and in skin undergoing wound healingdesignated as granulation tissue. Immunohistochemistry with antibodiesto β₃ and β₁ was performed as described in Example 3A. FIGS. 1A and 1Brespectively illustrate the immunoreactivity of anti-β₃ in normal skinand granulation tissue. FIGS. 1C and 1D respectively illustrate theimmunoreactivity of anti-β₁ in normal skin and granulation tissue.

FIGS. 2A-2D illustrate the tissue distribution of the von Willebrandfactor and laminin ligands that respectively bind the β₃ and β₁ integrinsubunits in normal skin and in skin undergoing wound healing designatedas granulation tissue. Immunohistochemistry with antibodies to vonWillebrand factor (anti-vWF) and laminin (anti-laminin) was performed asdescribed in Example 3B. FIGS. 2A and 2B respectively illustrate theimmunoreactivity of anti-vWF in normal skin and granulation tissue.FIGS. 2C and 2D respectively illustrate the immunoreactivity ofanti-laminin in normal skin and granulation tissue.

FIGS. 3A-3D illustrate the tissue distribution of the vitronectinintegrin receptor, α_(v)β₃, in tissue biopsies of bladder cancer, coloncancer, breast cancer and lung cancer, respectively.Immunohistochemistry with the LM609 antibody against α_(v)β₃ wasperformed as described in Example 3C.

FIG. 4 illustrates a typical photomicrograph of a CAM of this inventiondevoid of blood vessels in an untreated 10 day old chick embryo. Thepreparation is described in Example 5B.

FIGS. 5A-5C illustrate the tissue distribution of the integrins β₁ andα_(v)β₃ in the CAM preparation of this invention. FIG. 5A shows thedistribution of the β₁ subunit in an untreated 10 day old CAMpreparation as detected by immunofluorescence immunoreactivity withCSAT, an anti-β₁ antibody. FIG. 5B shows the distribution of the α_(v)β₃receptor in an untreated 10 day old CAM preparation as detected byimmunofluorescence immunoreactivity with LM609, an anti-α_(v)β₃antibody. FIG. 5C shows the distribution of the α_(v)β₃ receptor in anβFGF treated 10 day old CAM preparation as detected byimmunofluorescence immunoreactivity with LM609, an anti-α_(v)β₃antibody. The treatments and results are described in Example 5C.

FIG. 6 illustrates the quantification in a bar graph of the relativeexpression of α_(v)β₃ and β₁ in untreated and βFGF treated 10 day oldCAMs as described in Example 6A. The mean fluorescence intensity isplotted on the Y-axis with the integrin profiles plotted on the X-axis.

FIGS. 7A-7C illustrates the appearance of an untreated 10 day old CAM, aβFGF treated CAM, and a TNFα treated CAM, respectively, the proceduresand results of which are described in Example 6A.

FIGS. 8A-8E illustrate the effect of topical antibody treatment onFGF-induced angiogenesis in a day 10 CAM as described in Example 7A1).FIG. 8A shows an untreated CAM preparation that is devoid of bloodvessels. FIG. 8B shows the infiltration of new vasculature into an areapreviously devoid of vasculature induced by βFGF treatment. FIGS. 8C, 8Dand 8E respectively show the effects of antibodies against β₁ (anti-β₁;CSAT), α_(v)β₅ (anti-α_(v)B %; P3G2) and α_(v)β₃ (anti-α_(v)β₃; LM609).

FIGS. 9A-9C illustrate the effect of intravenous injection of syntheticpeptide 66203 on angiogenesis induced by tumors as described in Example7D2). FIG. 9A shows the lack of inhibitory effect of intravenoustreatment with a control peptide (control peptide tumor) on angiogenesisresulting from tumor induction. The inhibition of such angiogenesis byintravenous injection of peptide 66203 (cyclic RGD tumor) is shown inFIG. 9B. The lack of inhibitory effects or cytotoxicity on maturepreexisting vessels following intravenous infusion of peptide 66203 inan are a adjacent to the tumor-treated area is shown in FIG. 9C (cyclicRGD adjacent CAM).

FIGS. 10A-10C illustrate the effect of intravenous application ofmonoclonal antibodies to growth factor induced angiogenesis as describedin Example 7B₁). FIG. 10A shows βFGF-induced angiogenesis not exposed toantibody treatment (control). No inhibition of angiogenesis resultedwhen a similar preparation was treated with anti-α_(v)β₅ antibody P3G2as shown in FIG. 10B. Inhibition of angiogenesis resulted with treatmentof anti-α_(v)β₃ antibody LM609 as shown in FIG. 10C.

FIGS. 11A and 11C illustrate the effect on embryonic angiogenesisfollowing topical application of anti-integrin antibodies as describedin Example 7C. Angiogenesis was not inhibited by treatment of a 6 dayCAM with anti-β₁, and anti-α_(v)β₅ antibodies respectively shown inFIGS. 11A and 11B. In contrast, treatment with the anti-α_(v)β₃ antibodyLM609 resulted in the inhibition of blood vessel formation as shown inFIG. 11C.

FIG. 12 illustrates the quantification of the number of vessels enteringa tumor in a CAM preparation as described in Example 7D1). The graphshows the number of vessels as plotted on the Y-axis resulting fromtopical application of either CSAT (anti-β₁), LM609 (anti-α_(v)β₃) orP3G2 (anti-α_(v)β₅).

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

Amino Acid Residue: An amino acid formed upon chemical digestion(hydrolysis) of a polypeptide at its peptide linkages. The amino acidresidues described herein are preferably in the “L” isomeric form.However, residues in the “D” isomeric form can be substituted for anyL-amino acid residue, as long as the desired functional property isretained by the polypeptide. NH₂ refers to the free amino group presentat the amino terminus of a polypeptide. COOH refers to the free carboxygroup present at the carboxy terminus of a polypeptide. In keeping withstandard polypeptide nomenclature (described in J. Biol. Chem.,243:3552-59 (1969) and adopted at 37 CFR §1.822 (b) (2)), abbreviationsfor amino acid residues are shown in the following Table ofCorrespondence:

TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyrtyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGlu glutamic acid Z Glx Glu and/or Gln W Trp tryptophan R Arg arginine DAsp aspartic acid N Asn asparagine B Asx Asn and/or Asp C Cys cysteine XXaa Unknown or otherIn addition the following have the meanings below:

BOC tert-butyloxycarbonyl DCCI dicylcohexylcarbodiimide DMFdimethylformamide OMe methoxy HOBt 1-hydroxybezotriazole

It should be noted that all amino acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino acid residues.

Polypeptide: refers to a linear series of amino acid residues connectedto one-another by peptide bonds between the alpha-amino group andcarboxy group of contiguous amino acid residues.

Peptide: as used herein refers to a linear series of no more than about50 amino acid residues connected one to the other as in a polypeptide.

Cyclic peptide: is derived from a corresponding linear peptide and;refers to a peptide in which no free N- or C-termini exist and; and ofwhich the corresponding linear peptide's N-termini forms an amide bondto the C-terminal carboxylate of the said corresponding linear peptide.

Protein: refers to a linear series of greater than 50 amino acidresidues connected one to the other as in a polypeptide.

Synthetic peptide: refers to a chemically produced chain of amino acidresidues linked together by peptide bonds that is free of naturallyoccurring proteins and fragments thereof.

B. General Considerations

The present invention relates generally to the discovery thatangiogenesis is mediated by the specific vitronectin receptor α_(v)β₃,and that inhibition of α_(v)β₃ function inhibits angiogenesis. Thisdiscovery is important because of the role that angiogenesis plays in avariety of disease processes. By inhibiting angiogenesis, one canintervene in the disease, ameliorate the symptoms, and in some casescure the disease.

Where the growth of new blood vessels is the cause of, or contributesto, the pathology associated with a disease, inhibition of angiogenesiswill reduce the deleterious effects of the disease. Examples includerheumatoid arthritis, diabetic retinopathy, and the like. Where thegrowth of new blood vessels is required to support growth of adeleterious tissue, inhibition of angiogenesis will reduce the bloodsupply to the tissue and thereby contribute to reduction in tissue massbased on blood supply requirements. Examples include growth of tumorswhere neovascularization is a continual requirement in order that thetumor grow beyond a few millimeters in thickness, and for theestablishment of solid tumor metastases.

The methods of the present invention are effective in part because thetherapy is highly selective for angiogenesis and not other biologicalprocesses. As shown in the Examples, only new vessel growth containssubstantial α_(v)β₃, and therefore the therapeutic methods do notadversely effect mature vessels. Furthermore, α_(v)β₃ is not widelydistributed in normal tissues, but rather is found selectively on newvessels, thereby assuring that the therapy can be selectively targeted.

The discovery that inhibition of α_(v)β₃ alone will effectively inhibitangiogenesis allows for the development of therapeutic compositions withpotentially high specificity, and therefore relatively low toxicity.Thus although the invention discloses the use of RGD-peptide-basedreagents which have the ability to inhibit one or more integrins, onecan design reagents which selectively inhibit α_(v)β₃, and therefore donot have the side effect of inhibiting other biological processes otherthat those mediated by α_(v)β₃.

As shown by the present teachings, it is possible to prepare monoclonalantibodies highly selective for immunoreaction with α_(v)β₃ that aresimilarly selective for inhibition of α_(v)β₃ function. In addition,RGD-containing peptides can be designed to be selective for inhibitionof α_(v)β₃, as described further herein.

Prior to the discoveries of the present invention, it was not known thatangiogenesis could be inhibited in vivo by the use of reagents thatantagonize the biological function of α_(v)β₃.

C. Methods for Inhibition of Angiogenesis

The invention provides for a method for the inhibition of angiogenesisin a tissue, and thereby inhibiting events in the tissue which dependupon angiogenesis. Generally, the method comprises administering to thetissue a composition comprising an angiogenesis-inhibiting amount of anα_(v)β₃ antagonist.

As described earlier, angiogenesis includes a variety of processesinvolving neovascularization of a tissue including “sprouting”,vasculogenesis, or vessel enlargement, all of which angiogenesisprocesses are mediated by and dependent upon the expression of α_(v)β₃.With the exception of traumatic wound healing, corpus leuteum formationand embryogenesis, it is believed that the majority of angiogenesisprocesses are associated with disease processes.

There are a variety of diseases in which angiogenesis is believed to beimportant, referred to as angiogenic diseases, including but not limitedto, inflammatory disorders such as immune and non-immune inflammation,chronic articular rheumatism and psoriasis, disorders associated withinappropriate or inopportune invasion of vessels such as diabeticretinopathy, neovascular glaucoma, capillary proliferation inatherosclerotic plaques and osteoporosis, and cancer associateddisorders, such as solid tumors, solid tumor metastases, angiofibromas,retrolental fibroplasia, hemangiomas, Karposi sarcoma and the likecancers which require neovascularization to support tumor growth.

Thus, methods which inhibit angiogenesis in a diseased tissueameliorates symptoms of the disease and, depending upon the disease, cancontribute to cure of the disease. In one embodiment, the inventioncontemplates inhibition of angiogenesis, per se, in a tissue. The extentof angiogenesis in a tissue, and therefore the extent of inhibitionachieved by the present methods, can be evaluated by a variety ofmethods, such as are described in the Examples for detectingα_(v)β₃-immunopositive immature and nascent vessel structures byimmunohistochemistry.

As described herein, any of a variety of tissues, or organs comprised oforganized tissues, can support angiogenesis in disease conditionsincluding skin, muscle, gut, connective tissue, joints, bones and thelike tissue in which blood vessels can invade upon angiogenic stimuli.

Thus, in one related embodiment, a tissue to be treated is an inflamedtissue and the angiogenesis to be inhibited is inflamed tissueangiogenesis where there is neovascularization of inflamed tissue. Inthis class the method contemplates inhibition of angiogenesis inarthritic tissues, such as in a patient with chronic articularrheumatism, in immune or non-immune inflamed tissues, in psoriatictissue and the like.

The patient treated in the present invention in its many embodiments isdesirably a human patient, although it is to be understood that theprinciples of the invention indicate that the invention is effectivewith respect to all mammals, which are intended to be included in theterm “patient”. In this context, a mammal is understood to include anymammalian species in which treatment of diseases associated withangiogenesis is desirable, particularly agricultural and domesticmammalian species.

In another related embodiment, a tissue to be treated is a retinaltissue of a patient with diabetic retinopathy, macular degeneration orneovascular glaucoma and the angiogenesis to be inhibited is retinaltissue angiogenesis where there is neovascularization of retinal tissue.

In an additional related embodiment, a tissue to be treated is a tumortissue of a patient with a solid tumor, a metastases, a skin cancer, ahemangioma or angiofibroma and the like cancer, and the angiogenesis tobe inhibited is tumor tissue angiogenesis where there isneovascularization of a tumor tissue. Exemplary tumor tissueangiogenesis, and inhibition thereof, is described in the Examples.

Inhibition of tumor tissue angiogenesis is a particularly preferredembodiment because of the important role neovascularization plays intumor growth. In the absence of neovascularization of tumor tissue, thetumor tissue does not obtain the required nutrients, slows in growth,ceases additional growth, regresses and ultimately becomes necroticresulting in killing of the tumor.

Stated in other words, the present invention provides for a method ofinhibiting tumor neovascularization by inhibiting tumor angiogenesisaccording to the present methods. Similarly, the invention provides amethod of inhibiting tumor growth by practicing theangiogenesis-inhibiting methods.

The methods are also particularly effective against the formation ofmetastases because (1) their formation requires vascularization of aprimary tumor so that the metastatic cancer cells can exit the primarytumor and (2) their establishment in a secondary site requiresneovascularization to support growth of the metastases.

In a related embodiment, the invention contemplates the practice of themethod in conjunction with other therapies such as conventionalchemotherapy directed against solid tumors and for control ofestablishment of metastases. The administration of angiogenesisinhibitor is typically conducted during or after chemotherapy, althoughit is preferably to inhibit angiogenesis after a regimen of chemotherapyat times where the tumor tissue will be responding to the toxic assaultby inducing angiogenesis to recover by the provision of a blood supplyand nutrients to the tumor tissue. In addition, it is preferred toadminister the angiogenesis inhibition methods after surgery where solidtumors have been removed as a prophylaxis against metastases.

The present method for inhibiting angiogenesis in a tissue comprisescontacting a tissue in which angiogenesis is occurring, or is at riskfor occurring, with a composition comprising a therapeutically effectiveamount of an α_(v)β₃ antagonist capable of inhibiting α_(v)β₃ binding toits natural ligand. Thus the method comprises administering to a patienta therapeutically effective amount of a physiologically tolerablecomposition containing an α_(v)β₃ antagonist of the invention.

The dosage ranges for the administration of the α_(v)β₃ antagonistdepend upon the form of the antagonist, and its potency, as describedfurther herein, and are amounts large enough to produce the desiredeffect in which angiogenesis and the disease symptoms mediated byangiogenesis are ameliorated. The dosage should not be so large as tocause adverse side effects, such as hyperviscosity syndromes, pulmonaryedema, congestive heart failure, and the like. Generally, the dosagewill vary with the age, condition, sex and extent of the disease in thepatient and can be determined by one of skill in the art. The dosage canalso be adjusted by the individual physician in the event of anycomplication.

A therapeutically effective amount is an amount of α_(v)β₃ antagonistsufficient to produce a measurable inhibition of angiogenesis in thetissue being treated, ie., and angiogenesis-inhibiting amount.Inhibition of angiogenesis can be measured in situ byimmunohistochemistry, as described herein, or by other methods known toone skilled in the art.

Insofar as an α_(v)β₃ antagonist can take the form of α_(v)β₃ mimetic,and RGD-containing peptide, an anti-α_(v)β₃ monoclonal antibody, orfragment thereof, it is to be appreciated that the potency, andtherefore an expression of a “therapeutically effective” amount canvary. However, as shown by the present assay methods, one skilled in theart can readily assess the potency of a candidate α_(v)β₃ antagonist ofthis invention.

Potency of an α_(v)β₃ antagonist can be measured by a variety of meansincluding inhibition of angiogenesis in the CAM assay described herein,inhibition of binding of natural ligand to α_(v)β₃ as described herein,and the like assays.

A preferred α_(v)β₃ antagonist has the ability to substantially inhibitbinding of a natural ligand such as fibrinogen or vitronectin to α_(v)β₃in solution at antagonist concentrations of less than 0.5 micromolar(uM), preferably less than 0.1 uM, and more preferably less than 0.05uM. By “substantially” is meant that at least a 50 percent reduction inbinding of fibrinogen is observed by inhibition in the presence of theα_(v)β₃ antagonist, and at 50% inhibition is referred to herein as anIC₅₀ value.

A more preferred α_(v)β₃ antagonist exhibits selectivity for α_(v)β₃over other integrins. Thus, a preferred α_(v)β₃ antagonist substantiallyinhibits fibrinogen binding to α_(v)β₃ but does not substantiallyinhibit binding of fibrinogen to another integrin, such as α_(v)β₁,α_(v)β₅ or α_(IIb)β₃. Particularly preferred is an α_(v)β₃ antagonistthat exhibits a 10-fold to 100-fold lower IC₅₀ activity at inhibitingfibrinogen binding to α_(v)β₃ compared to the IC₅₀ activity atinhibiting fibrinogen binding to another integrin. Exemplary assays formeasuring IC₅₀ activity at inhibiting fibrinogen binding to an integrinare described in the Examples.

A therapeutically effective amount of an α_(v)β₃ antagonist of thisinvention in the form of a monoclonal antibody, or fragment thereof, istypically an amount such that when administered in a physiologicallytolerable composition is sufficient to achieve a plasma concentration offrom about 0.01 microgram (ug) per milliliter (ml) to about 100 ug/ml,preferably from about 1 ug/ml to about 5 ug/ml, and usually about 5ug/ml. Stated differently, the dosage can vary from about 0.1 mg/kg toabout 300 mg/kg, preferably from about 0.2 mg/kg to about 200 mg/kg,most preferably from about 0.5 mg/kg to about 20 mg/kg, in one or moredose administrations daily, for one or several days.

A therapeutically effective amount of an α_(v)β₃ antagonist of thisinvention in the form of a polypeptide is typically an amount ofpolypeptide such that when administered in a physiologically tolerablecomposition is sufficient to achieve a plasma concentration of fromabout 0.1 microgram (ug) per milliliter (ml) to about 200 ug/ml,preferably from about 1 ug/ml to about 150 ug/ml. Based on a polypeptidehaving a mass of about 500 grams per mole, the preferred plasmaconcentration in molarity is from about 2 micromolar (uM) to about 5millimolar (mM) and preferably about 100 uM to 1 mM polypeptideantagonist. Stated differently, the dosage per body weight can vary fromabout 0.1 mg/kg to about 300 mg/kg, and preferably from about 0.2 mg/kgto about 200 mg/kg, in one or more dose administrations daily, for oneor several days.

The monoclonal antibodies or polypeptides of the invention can beadministered parenterally by injection or by gradual infusion over time.Although the tissue to be treated can typically be accessed in the bodyby systemic administration and therefore most often treated byintravenous administration of therapeutic compositions, other tissuesand delivery means are contemplated where there is a likelihood that thetissue targeted contains the target molecule. Thus, monoclonalantibodies or polypeptides of the invention can be administeredintravenously, intraperitoneally, intramuscularly, subcutaneously,intracavity, transdermally, and can be delivered by peristaltic means.

The therapeutic compositions containing a monoclonal antibody or apolypeptide of this invention are conventionally administeredintravenously, as by injection of a unit dose, for example. The term“unit dose” when used in reference to a therapeutic composition of thepresent invention refers to physically discrete units suitable asunitary dosage for the subject, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect in association with the required diluent; i.e.,carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's system to utilize the active ingredient, and degree oftherapeutic effect desired. Precise amounts of active ingredientrequired to be administered depend on the judgement of the practitionerand are peculiar to each individual. However, suitable dosage ranges forsystemic application are disclosed herein and depend on the route ofadministration. Suitable regimes for administration are also variable,but are typified by an initial administration followed by repeated dosesat one or more hour intervals by a subsequent injection or otheradministration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations in the blood in the rangesspecified for in vivo therapies are contemplated.

D. Therapeutic Compositions

The present invention contemplates therapeutic compositions useful forpracticing the therapeutic methods described herein. Therapeuticcompositions of the present invention contain a physiologicallytolerable carrier together with an α_(v)β₃ antagonist as describedherein, dissolved or dispersed therein as an active ingredient. In apreferred embodiment, the therapeutic α_(v)β₃ antagonist composition isnot immunogenic when administered to a mammal or human patient fortherapeutic purposes.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a mammal without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset and thelike.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in the artand need not be limited based on formulation. Typically suchcompositions are prepared as injectables either as liquid solutions orsuspensions, however, solid forms suitable for solution, or suspensions,in liquid prior to use can also be prepared. The preparation can also beemulsified.

The active ingredient can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredientand in amounts suitable for use in the therapeutic methods describedherein. Suitable excipients are, for example, water, saline, dextrose,glycerol, ethanol or the like and combinations thereof. In addition, ifdesired, the composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like which enhance the effectiveness of the active ingredient.

The therapeutic composition of the present invention can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Particularly preferred are the salts of TFA and HCl, when used in thepreparation of cyclic polypeptide α_(v)β₃ antagonists. Representativesalts of peptides are described in the Examples.

Physiologically tolerable carriers are well known in the art. Exemplaryof liquid carriers are sterile aqueous solutions that contain nomaterials in addition to the active ingredients and water, or contain abuffer such as sodium phosphate at physiological pH value, physiologicalsaline or both, such as phosphate-buffered saline. Still further,aqueous carriers can contain more than one buffer salt, as well as saltssuch as sodium and potassium chlorides, dextrose, polyethylene glycoland other solutes.

Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil, and water-oilemulsions.

A therapeutic composition contains an angiogenesis-inhibiting amount ofan α_(v)β₃ antagonist of the present invention, typically formulated tocontain an amount of at least 0.1 weight percent of antagonist perweight of total therapeutic composition. A weight percent is a ratio byweight of inhibitor to total composition. Thus, for example, 0.1 weightpercent is 0.1 grams of inhibitor per 100 grams of total composition.

E. Antagonists of Integrin α_(v)β₃

α_(v)β₃ antagonists are used in the present methods for inhibitingangiogenesis in tissues, and can take a variety of forms that includecompounds which interact with α_(v)β₃ in a manner such that functionalinteractions with natural α_(v)β₃ ligands are interfered. Exemplaryantagonists include analogs of α_(v)β₃ derived from the ligand bindingsite on α_(v)β₃, mimetics of either α_(v)β₃ or a natural ligand ofα_(v)β₃ that mimic the structural region involved in α_(v)β₃-ligandbinding interactions, polypeptides having a sequence corresponding tothe RGD-containing domain of a natural ligand of α_(v)β₃, and antibodieswhich immunoreact with either α_(v)β₃ or the natural ligand, all ofwhich exhibit antagonist activity as defined herein.

1. Polypeptides

In one embodiment, the invention contemplates α_(v)β₃ antagonists in theform of polypeptides. A polypeptide (peptide) α_(v)β₃ antagonist canhave the sequence characteristics of either the natural ligand ofα_(v)β₃ or α_(v)β₃ itself at the region involved in α_(v)β₃-ligandinteraction and exhibits α_(v)β₃ antagonist activity as describedherein. A preferred α_(v)β₃ antagonist peptide contains the RGDtripeptide and corresponds in sequence to the natural ligand in theRGD-containing region.

Preferred RGD-containing polypeptides have a sequence corresponding tothe amino acid residue sequence of the RGD-containing region of anatural ligand of α_(v)β₃ such as fibrinogen, vitronectin, vonWillebrand factor, laminin, thrombospondin, and the like ligands. Thesequence of these α_(v)β₃ ligands are well known. Thus, an α_(v)β₃antagonist peptide can be derived from any of the natural ligands,although fibrinogen and vitronectin are preferred.

A particularly preferred α_(v)β₃ antagonist peptide preferentiallyinhibits α_(v)β₃ binding to its natural ligand(s) when compared to otherintegrins, as described earlier. These α_(v)β₃-specific peptides areparticularly preferred at least because the specificity for α_(v)β₃reduces the incidence of undesirable side effects such as inhibition ofother integrins. The identification of preferred α_(v)β₃ antagonistpeptides having selectivity for α_(v)β₃ can readily be identified in atypical inhibition of binding assay, such as the ELISA assay describedin the Examples.

In one embodiment, a polypeptide of the present invention comprises nomore than about 100 amino acid residues, preferably no more than about60 residues, more preferably no more than about 30 residues. Peptidescan be linear or cyclic, although particularly preferred peptides arecyclic.

Preferred cyclic and linear peptides and their designations are shown inTable 1 in the Examples.

It should be understood that a subject polypeptide need not be identicalto the amino acid residue sequence of α_(v)β₃ natural ligand, so long asit includes the required sequence and is able to function as an α_(v)β₃antagonist in an assay such as is described herein.

A subject polypeptide includes any analog, fragment or chemicalderivative of a polypeptide whose amino acid residue sequence is shownherein so long as the polypeptide is an α_(v)β₃ antagonist. Therefore, apresent polypeptide can be subject to various changes, substitutions,insertions, and deletions where such changes provide for certainadvantages in its use. In this regard, α_(v)β₃ antagonist polypeptide ofthis invention corresponds to, rather than is identical to, the sequenceof a recited peptide where one or more changes are made and it retainsthe ability to function as an α_(v)β₃ antagonist in one or more of theassays as defined herein.

Thus, a polypeptide can be in any of a variety of forms of peptidederivatives, that include amides, conjugates with proteins, cyclizedpeptides, polymerized peptides, analogs, fragments, chemically modifiedpeptides, and the like derivatives.

The term “analog” includes any polypeptide having an amino acid residuesequence substantially identical to a sequence specifically shown hereinin which one or more residues have been conservatively substituted witha functionally similar residue and which displays the α_(v)β₃ antagonistactivity as described herein. Examples of conservative substitutionsinclude the substitution of one non-polar (hydrophobic) residue such asisoleucine, valine, leucine or methionine for another, the substitutionof one polar (hydrophilic) residue for another such as between arginineand lysine, between glutamine and asparagine, between glycine andserine, the substitution of one basic residue such as lysine, arginineor histidine for another, or the substitution of one acidic residue,such as aspartic acid or glutamic acid for another.

The phrase “conservative substitution” also includes the use of achemically derivatized residue in place of a non-derivatized residueprovided that such polypeptide displays the requisite inhibitionactivity.

“Chemical derivative” refers to a subject polypeptide having one or moreresidues chemically derivatized by reaction of a functional side group.Such derivatized molecules include for example, those molecules in whichfree-amino groups have been derivatized to form amine hydrochlorides,p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonylgroups, chloroacetyl groups or formyl groups. Free carboxyl groups maybe derivatized to form salts, methyl and ethyl esters or other types ofesters or hydrazides. Free hydroxyl groups may be derivatized to formO-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine maybe derivatized to form N-im-benzylhistidine. Also included as chemicalderivatives are those peptides which contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids. Forexamples: 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine. Polypeptides of the presentinvention also include any polypeptide having one or more additionsand/or deletions or residues relative to the sequence of a polypeptidewhose sequence is shown herein, so long as the requisite activity ismaintained.

The term “fragment” refers to any subject polypeptide having an aminoacid residue sequence shorter than that of a polypeptide whose aminoacid residue sequence is shown herein.

When a polypeptide of the present invention has a sequence that is notidentical to the sequence of an α_(v)β₃ natural ligand, it is typicallybecause one or more conservative or non-conservative substitutions havebeen made, usually no more than about 30 number percent, and preferablyno more than 10 number percent of the amino acid residues aresubstituted. Additional residues may also be added at either terminus ofa polypeptide for the purpose of providing a “linker” by which thepolypeptides of this invention can be conveniently affixed to a label orsolid matrix, or carrier.

Labels, solid matrices and carriers that can be used with thepolypeptides of this invention are described herein below.

Amino acid residue linkers are usually at least one residue and can be40 or more residues, more often 1 to 10 residues, but do not formα_(v)β₃ ligand epitopes. Typical amino acid residues used for linkingare tyrosine, cysteine, lysine, glutamic and aspartic acid, or the like.In addition, a subject polypeptide can differ, unless otherwisespecified, from the natural sequence of α_(v)β₃ ligand by the sequencebeing modified by terminal-NH₂ acylation, e.g., acetylation, orthioglycolic acid amidation, by terminal-carboxylamidation, e.g., withammonia, methylamine, and the like terminal modifications. Terminalmodifications are useful, as is well known, to reduce susceptibility byproteinase digestion, and therefore serve to prolong half life of thepolypeptides in solutions, particularly biological fluids whereproteases may be present. In this regard, polypeptide cyclization isalso a useful terminal modification, and is particularly preferred alsobecause of the stable structures formed by cyclization and in view ofthe biological activities observed for such cyclic peptides as describedherein.

Any peptide of the present invention may be used in the form of apharmaceutically acceptable salt. Suitable acids which are capable offorming salts with the peptides of the present invention includeinorganic acids such as trifluoroacetic acid (TFA) hydrochloric acid(HCl), hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,sulfuric acid, phosphoric acetic acid, propionic acid, glycolic acid,lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalenesulfonic acid, sulfanilic acid or the like. HCl and TFA salts areparticularly preferred.

Suitable bases capable of forming salts with the peptides of the presentinvention include inorganic bases such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide and the like; and organic bases such asmono-, di- and tri-alkyl and aryl amines (e.g. triethylamine,diisopropyl amine, methyl amine, dimethyl amine and the like) andoptionally substituted ethanolamines (e.g. ethanolamine, diethanolamineand the like).

A peptide of the present invention also referred to herein as a subjectpolypeptide, can be synthesized by any of the techniques that are knownto those skilled in the polypeptide art, including recombinant DNAtechniques. Synthetic chemistry techniques, such as a solid-phaseMerrifield-type synthesis, are preferred for reasons of purity,antigenic specificity, freedom from undesired side products, ease ofproduction and the like. An excellent summary of the many techniquesavailable can be found in Steward et al., “Solid Phase PeptideSynthesis”, W. H. Freeman Co., San Francisco, 1969; Bodanszky, et al.,“Peptide Synthesis”, John Wiley & Sons, Second Edition, 1976; J.Meienhofer, “Hormonal Proteins and Peptides”, Vol. 2, p. 46, AcademicPress (New York), 1983; Merrifield, Adv. Enzymol., 32:221-96, 1969;Fields et al., int. J. Peptide Protein Res., 35:161-214, 1990; and U.S.Pat. No. 4,244,946 for solid phase peptide synthesis, and Schroder etal., “The Peptides”, Vol. 1, Academic Press (New York), 1965 forclassical solution synthesis, each of which is incorporated herein byreference. Appropriate protective groups usable in such synthesis aredescribed in the above texts and in J. F. W. McOmie, “Protective Groupsin Organic Chemistry”, Plenum Press, New York, 1973, which isincorporated herein by reference.

In general, the solid-phase synthesis methods contemplated comprise thesequential addition of one or more amino acid residues or suitablyprotected amino acid residues to a growing peptide chain. Normally,either the amino or carboxyl group of the first amino acid residue isprotected by a suitable, selectively removable protecting group. Adifferent, selectively removable protecting group is utilized for aminoacids containing a reactive side group such as lysine.

Using a solid phase synthesis as exemplary, the protected or derivatizedamino acid is attached to an inert solid support through its unprotectedcarboxyl or amino group. The protecting group of the amino or carboxylgroup is then selectively removed and the next amino acid in thesequence having the complimentary (amino or carboxyl) group suitablyprotected is admixed and reacted under conditions suitable for formingthe amide linkage with the residue already attached to the solidsupport. The protecting group of the amino or carboxyl group is thenremoved from this newly added amino acid residue, and the next aminoacid (suitably protected) is then added, and so forth. After all thedesired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to afford the final linearpolypeptide.

The resultant linear polypeptides prepared for example as describedabove may be reacted to form their corresponding cyclic peptides. Anexemplary method for cyclizing peptides is described by Zimmer et al.,Peptides 1992, pp. 393-394, ESCOM Science Publishers, B. V., 1993.Typically, tertbutoxycarbonyl protected peptide methyl ester isdissolved in methanol and sodium hydroxide solution are added and theadmixture is reacted at 20° C. (20 C) to hydrolytically remove themethyl ester protecting group. After evaporating the solvent, thetertbutoxycarbonyl protected peptide is extracted with ethyl acetatefrom acidified aqueous solvent. The tertbutoxycarbonyl protecting groupis then removed under mildly acidic conditions in dioxane cosolvent. Theunprotected linear peptide with free amino and carboxy termini soobtained is converted to its corresponding cyclic peptide by reacting adilute solution of the linear peptide, in a mixture of dichloromethaneand dimethylformamide, with dicyclohexylcarbodiimide in the presence of1-hydroxybenzotriazole and N-methylmorpholine. The resultant cyclicpeptide is then purified by chromatography.

A particularly preferred cyclic peptide synthesis method is described byGurrath et al., Eur. J. Biochem., 210:911-921 (1992), and described inthe Examples. Particularly preferred peptides for use in the presentmethods are c-(GrGDFV) (SEQ ID NO 4), c-(RGDfV) (SEQ ID NO 5), c-(RADfV)(SEQ ID NO 6), c-(RGDFv) (SEQ ID NO 7) and linear peptideYTAECKPQVTRGDVF (SEQ ID NO 8), where “c-” indicates a cyclic peptide,the upper case letters are single letter code for an L-amino acid andthe lower case letters are single letter code for D-amino acid. Theamino acid residues sequence of these peptides are also shown in SEQ. IDNOs 4, 5, 6, 7 and 8, respectively.

2. Monoclonal Antibodies

The present invention describes, in one embodiment, α_(v)β₃ antagonistsin the form of monoclonal antibodies which immunoreact with α_(v)β₃ andinhibit α_(v)β₃ binding to its natural ligand as described herein. Theinvention also describes cell lines which produce the antibodies,methods for producing the cell lines, and methods for producing themonoclonal antibodies.

A monoclonal antibody of this invention comprises antibody moleculesthat 1) immunoreact with isolated α_(v)β₃, and 2) inhibit fibrinogenbinding to α_(v)β₃. Preferred monoclonal antibodies which preferentiallybind to α_(v)β₃ include a monoclonal antibody having the immunoreactioncharacteristics of Mab LM609, secreted by hybridoma cell line ATCC HB9537. The hybridoma cell line ATCC HB 9537 was deposited pursuant toBudapest Treaty requirements with the American Type Culture Collection(ATCC), 1301 Parklawn Drive, Rockville, Md., USA, on Sep. 15, 1987.

The term “antibody or antibody molecule” in the various grammaticalforms is used herein as a collective noun that refers to a population ofimmunoglobulin molecules and/or immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antibodycombining site or paratope.

An “antibody combining site” is that structural portion of an antibodymolecule comprised of heavy and light chain variable and hypervariableregions that specifically binds antigen.

Exemplary antibodies for use in the present invention are intactimmunoglobulin molecules, substantially intact immunoglobulin moleculesand those portions of an immunoglobulin molecule that contain theparatope, including those portions known in the art as Fab, Fab′, F(ab′)₂ and F(v), and also referred to as antibody fragments.

In another preferred embodiment, the invention contemplates a truncatedimmunoglobulin molecule comprising a Fab fragment derived from amonoclonal antibody of this invention. The Fab fragment, lacking Fcreceptor, is soluble, and affords therapeutic advantages in serum halflife, and diagnostic advantages in modes of using the soluble Fabfragment. The preparation of a soluble Fab fragment is generally knownin the immunological arts and can be accomplished by a variety ofmethods.

For example, Fab and F(ab′)₂ portions (fragments) of antibodies areprepared by the proteolytic reaction of papain and pepsin, respectively,on substantially intact antibodies by methods that are well known. Seefor example, U.S. Pat. No. 4,342,566 to Theofilopolous and Dixon. Fab′antibody portions are also well known and are produced from F(ab′)₂portions followed by reduction of the disulfide bonds linking the twoheavy chain portions as with mercaptoethanol, and followed by alkylationof the resulting protein mercaptan with a reagent such as iodoacetamide.An antibody containing intact immunoglobulin molecules are preferred,and are utilized as illustrative herein.

The phrase “monoclonal antibody” in its various grammatical forms refersto a population of antibody molecules that contain only one species ofantibody combining site capable of immunoreacting with a particularepitope. A monoclonal antibody thus typically displays a single bindingaffinity for any epitope with which it immunoreacts. A monoclonalantibody may therefore contain an antibody molecule having a pluralityof antibody combining sites, each immunospecific for a differentepitope, e.g., a bispecific monoclonal antibody.

A monoclonal antibody is typically composed of antibodies produced byclones of a single cell called a hybridoma that secretes (produces) onlyone kind of antibody molecule. The hybridoma cell is formed by fusing anantibody-producing cell and a myeloma or other self-perpetuating cellline. The preparation of such antibodies was first described by Kohlerand Milstein, Nature 256:495-497 (1975), which description isincorporated by reference. Additional methods are described by Zola,Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987).The hybridoma supernates so prepared can be screened for the presence ofantibody molecules that immunoreact with α_(v)β₃ and for inhibition ofα_(v)β₃ binding to natural ligands.

Briefly, to form the hybridoma from which the monoclonal antibodycomposition is produced, a myeloma or other self-perpetuating cell lineis fused with lymphocytes obtained from the spleen of a mammalhyperimmunized with a source of α_(v)β₃, such as α_(v)β₃ isolated fromM21 human melanoma cells as described by Cheresh et al., J. Biol. Chem.,262:17703-17711 (1987).

It is preferred that the myeloma cell line used to prepare a hybridomabe from the same species as the lymphocytes. Typically, a mouse of thestrain 129 GlX⁺ is the preferred mammal. Suitable mouse myelomas for usein the present invention include thehypoxanthine-aminopterin-thymidine-sensitive (HAT) cell linesP3X63-Ag8.653, and Sp2/0-Ag14 that are available from the American TypeCulture Collection, Rockville, Md., under the designations CRL 1580 andCRL 1581, respectively.

Splenocytes are typically fused with myeloma cells using polyethyleneglycol (PEG) 1500. Fused hybrids are selected by their sensitivity toHAT. Hybridomas producing a monoclonal antibody of this invention areidentified using the enzyme linked immunosorbent assay (ELISA) describedin the Examples.

A monoclonal antibody of the present invention can also be produced byinitiating a monoclonal hybridoma culture comprising a nutrient mediumcontaining a hybridoma that secretes antibody molecules of theappropriate specificity. The culture is maintained under conditions andfor a time period sufficient for the hybridoma to secrete the antibodymolecules into the medium. The antibody-containing medium is thencollected. The antibody molecules can then be further isolated by wellknown techniques.

Media useful for the preparation of these compositions are both wellknown in the art and commercially available and include syntheticculture media, inbred mice and the like. An exemplary synthetic mediumis Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol.8:396 (1959)) supplemented with 4.5 gm/1 glucose, 20 mM glutamine, and20% fetal calf serum. An exemplary inbred mouse strain is the Balb/c.

Other methods of producing a monoclonal antibody, a hybridoma cell, or ahybridoma cell culture are also well known. See, for example, the methodof isolating monoclonal antibodies from an immunological repertoire asdescribed by Sastry, et al., Proc. Natl. Acad. Sci. USA, 86:5728-5732(1989); and Huse et al., Science, 246:1275-1281 (1989).

Also contemplated by this invention is the hybridoma cell, and culturescontaining a hybridoma cell that produce a monoclonal antibody of thisinvention. Particularly preferred is the hybridoma cell line thatsecretes monoclonal antibody Mab LM609 designated ATCC HB 9537. MabLM609 was prepared as described by Cheresh et al., J. Biol. Chem.,262:17703-17711 (1987), and its preparation is also described in theExamples.

The invention contemplates, in one embodiment, a monoclonal antibodythat has the immunoreaction characteristics of Mab LM609.

It is also possible to determine, without undue experimentation, if amonoclonal antibody has the same (i.e., equivalent) specificity(immunoreaction characteristics) as a monoclonal antibody of thisinvention by ascertaining whether the former prevents the latter frombinding to a preselected target molecule. If the monoclonal antibodybeing tested competes with the monoclonal antibody of the invention, asshown by a decrease in binding by the monoclonal antibody of theinvention in standard competition assays for binding to the targetmolecule when present in the solid phase, then it is likely that the twomonoclonal antibodies bind to the same, or a closely related, epitope.

Still another way to determine whether a monoclonal antibody has thespecificity of a monoclonal antibody of the invention is to pre-incubatethe monoclonal antibody of the invention with the target molecule withwhich it is normally reactive, and then add the monoclonal antibodybeing tested to determine if the monoclonal antibody being tested isinhibited in its ability to bind the target molecule. If the monoclonalantibody being tested is inhibited then, in all likelihood, it has thesame, or functionally equivalent, epitopic specificity as the monoclonalantibody of the invention.

An additional way to determine whether a monoclonal antibody has thespecificity of a monoclonal antibody of the invention is to determinethe amino acid residue sequence of the CDR regions of the antibodies inquestion. Antibody molecules having identical, or functionallyequivalent, amino acid residue sequences in their CDR regions have thesame binding specificity. Methods for sequencing polypeptides is wellknown in the art.

The immunospecificity of an antibody, its target molecule bindingcapacity, and the attendant affinity the antibody exhibits for theepitope, are defined by the epitope with which the antibodyimmunoreacts. The epitope specificity is defined at least in part by theamino acid residue sequence of the variable region of the heavy chain ofthe immunoglobulin the antibody, and in part by the light chain variableregion amino acid residue sequence.

Use of the term “having the binding specificity of” indicates thatequivalent monoclonal antibodies exhibit the same or similarimmunoreaction (binding) characteristics and compete for binding to apreselected target molecule.

Humanized monoclonal antibodies offer particular advantages over murinemonoclonal antibodies, particularly insofar as they can be usedtherapeutically in humans. Specifically, human antibodies are notcleared from the circulation as rapidly as “foreign” antigens, and donot activate the immune system in the same manner as foreign antigensand foreign antibodies. Methods of preparing “humanized” antibodies aregenerally well known in the art, and can readily be applied to theantibodies of the present invention.

Thus, the invention contemplates, in one embodiment, a monoclonalantibody of this invention that is humanized by grafting to introducecomponents of the human immune system without substantially interferingwith the ability of the antibody to bind antigen.

F. Methods for Identifying Antagonists of α_(v)β₃

The invention also described assay methods for identifying candidateα_(v)β₃ antagonists for use according to the present methods. In theseassay methods candidate molecules are evaluated for their potency ininhibiting of α_(v)β₃ binding to natural ligands, and furthermore areevaluated for their potency in inhibiting angiogenesis in a tissue.

The first assay measures inhibition of direct binding of natural ligandto α_(v)β₃, and a preferred embodiment is described in detail in theExamples. The assay typically measures the degree of inhibition ofbinding of a natural ligand, such as fibrinogen, to isolated α_(v)β₃ inthe solid phase by ELISA.

The assay can also be used to identify compounds which exhibitspecificity for α_(v)β₃ and do not inhibit natural ligands from bindingother integrins. The specificity assay is conducted by running parallelELISA assays where both α_(v)β₃ and other integrins are screenedconcurrently in separate assay chambers for their respective abilitiesto bind a natural ligand and for the candidate compound to inhibit therespective abilities of the integrins to bind a preselected ligand.Preferred screening assay formats are described in the Examples.

The second assay measures angiogenesis in the chick chorioallantoicmembrane (CAM) and is referred to as the CAM assay. The CAM assay has bedescribed in detail by others, and further has been used to measure bothangiogenesis and neovascularization of tumor tissues. See Ausprunk etal., Am. J. Pathol., 79:597-618 (1975) and Ossonski et al., Cancer Res.,40:2300-2309 (1980).

The CAM assay is a well recognized assay model for in vivo angiogenesisbecause neovascularization of whole tissue is occurring, and actualchick embryo blood vessels are growing into the CAM or into the tissuegrown on the CAM.

As demonstrated herein, the CAM assay illustrates inhibition ofneovascularization based on both the amount and extent of new vesselgrowth. Furthermore, it is easy to monitor the growth of any tissuetransplanted upon the CAM, such as a tumor tissue. Finally, the assay isparticularly useful because there is an internal control for toxicity inthe assay system. The chick embryo is exposed to any test reagent, andtherefore the health of the embryo is an indication of toxicity.

EXAMPLES

The following examples relating to this invention are illustrative andshould not, of course, be construed as specifically limiting theinvention. Moreover, such variations of the invention, now known orlater developed, which would be within the purview of one skilled in theart are to be considered to fall within the scope of the presentinvention hereinafter claimed.

1. Preparation of Synthetic Peptides

The linear and cyclic polypeptides listed in Table 1 were synthesizedusing standard solid-phase synthesis techniques as, for example,described by Merrifield, Adv. Enzymol., 32:221-96, (1969), and Fields,G. B. and Noble, R. L., Int. J. Peptide Protein Res., 35:161-214,(1990).

Two grams (g) of BOC-Gly-D-Arg-Gly-Asp-Phe-Val-OMe (SEQ ID NO 1) werefirst dissolved in 60 milliliters (ml) of methanol to which was added1.5 ml of 2 N sodium hydroxide solution to form an admixture. Theadmixture was then stirred for 3 hours at 20 degrees C (20 C). Afterevaporation, the residue was taken up in water, acidified to pH 3 withdiluted HCl and extracted with ethyl acetate. The extract was dried overNa₂SO₄, evaporated again and the resultantBOC-Gly-D-Arg-Gly-Asp-Phe-Val-OH (SEQ ID NO 2) was stirred at 20 C for 2hours with 20 ml of 2 N HCl in dioxane. The resultant admixture wasevaporated to obtain H-Gly-D-Arg-Gly-Asp-Phe-Val-OH (SEQ ID NO 3) thatwas subsequently dissolved in a mixture of 1800 ml of dichloromethaneand 200 ml of dimethylformamide (DMF) followed by cooling to 0 C.Thereafter, 0.5 g of dicyclohexylcarbodiimide (DCCl), 0.3 g of1-hydroxybenzotriazole (HOBt) and 0.23 ml of N-methylmorpholine wereadded successively with stirring.

The resultant admixture was stirred for a further 24 hours at 0 C andthen at 20 C for another 48 hours. The solution was concentrated andtreated with a mixed bed ion exchanger to free it from salts. After theresulting resin was removed by filtration, the clarified solution wasevaporated and the residue was purified by chromatography resulting inthe recovery of cyclo(-Gly-D-Arg-Gly-Asp-Phe-Val) (SEQ ID NO 4). Thefollowing peptides, listed in Table 1 using single letter code aminoacid residue abbreviations and identified by a peptide numberdesignation, were obtained analogously: cyclo(Arg-Gly-Asp-D-Phe-Val)(SEQ ID NO 5); cyclo(Arg-Ala-Asp-D-Phe-Val) (SEQ ID NO 6);cyclo(Arg-D-Ala-Asp-Phe-Val) (SEQ ID NO 9); cyclo(Arg-Gly-Asp-Phe-D-Val)(SEQ ID NO 7). A peptide designated as 66203, having an identicalsequence to that of peptide 62184, only differed from the latter bycontaining the salt HCl rather than the TFA salt present in 62184. Ininhibition of angiogenesis assays as described in Example 7 where thesynthetic peptides were used, the 66203 peptide having HCl was slightlymore effective in inhibiting angiogenesis than the identical peptide inTFA.

TABLE 1 Peptide No. Amino Acid Sequence SEQ ID NO 62181 cyclo (GrGDFV) 4 62184 cyclo (RGDfV)  5 62185 cyclo (RADfV)  6 62187 cyclo (RGDFv)  762880 YTAECKPQVTRGDVF  8 62186 cyclo (RaDFV)  9 62175 cyclo (ARGDfL) 1062179 cyclo (GRGDfL) 11 62411 TRQVVCDLGNPM 12 62503 GVVRNNEALARLS 1362502 TDVNGDGRHDL 14 *Lower case letters indicate a D-amino acid;capital letters indicate a L-amino acid.2. Monoclonal Antibodies

The monoclonal antibody LM609 secreted by hybridoma ATCC HB 9537 wasproduced using standard hybridoma methods by immunization with isolatedα_(v)β₃ adsorbed onto SEPHAROSE-lentil lectin beads. The α_(v)β₃ hadbeen isolated from human melanoma cells designated M21, and antibody wasproduced as described by Cheresh et al., J. Biol. Chem., 262:17703-17711(1987). M21 cells were provided by Dr. D. L. Morton (University ofCalifornia at Los Angeles, Calif.) and grown in suspension cultures inRPMI 1640 culture medium containing 2 mM L-glutamine, 50 mg/mlgentamicin sulfate and 10% fetal calf serum.

Monoclonal antibody LM609 has been shown to immunoreact with α_(v)β₃complex, and not immunoreact with α_(v) subunit, with β₃ subunit, orwith other integrins.

3. Characterization of the Tissue Distribution of α_(v)β₃ Expression

A. Immunofluorescence with Anti-Integrin Receptor Antibodies

During wound healing, the basement membranes of blood vessels expressseveral adhesive proteins, including von Willebrand factor, fibronectin,and fibrin. In addition, several members of the integrin family ofadhesion receptors are expressed on the surface of cultured smoothmuscle and endothelial cells. See, Cheresh, Proc. Natl. Acad. Sci., USA,84:6471 (1987); Janat et al., J. Cell Physiol., 151:588 (1992); andCheng et al., J. Cell Physiol., 139:275 (1989). Among these integrins isα_(v)β₃, the endothelial cell receptor for von Willebrand factor,fibrinogen (fibrin), and fibronectin as described by Cheresh, Proc.Natl. Acad. Sci., USA, 84:6471 (1987). This integrin initiates acalcium-dependent signaling pathway leading to endothelial cellmigration, and therefore appears to play a fundamental role in vascularcell biology as described by Leavelsey et al., J. Cell Biol., 121:163(1993).

To investigate the expression of α_(v)β₃ during angiogenesis, humanwound granulation tissue or adjacent normal skin was obtained fromconsenting patients, washed with 1 ml of phosphate buffered saline andembedded in O.T.C medium (Tissue Tek). The embedded tissues were snapfrozen in liquid nitrogen for approximately 30 to 45 seconds. Six micronthick sections were cut from the frozen blocks on a cryostat microtomefor subsequent immunoperoxidase staining with antibodies specific foreither β₃ integrins (α_(v)β₃ or α_(IIb)β₃) or the β₃ subfamily ofintegrins.

The results of the staining of normal human skin and wound granulationtissue are shown in FIGS. 1A-1D. Monoclonal antibodies AP3 and LM534,directed to β₃ and β₃ integrins, respectively, were used forimmunohistochemical analysis of frozen sections. Experiments with tissuefrom four different human donors yielded identical results. Thephotomicrographs are shown at magnification of 300×.

The α_(v)β₃ integrin was abundantly expressed on blood vessels ingranulation tissue (FIG. 1B) but was not detectable in the dermis andepithelium of normal skin from the same donor (FIG. 1A). In contrast, β₁integrins were abundantly expressed on blood vessels and stromal cellsin both normal skin (FIG. 1C) and granulation tissue (FIG. 1D) and, aspreviously shown as described by Adams et al., Cell, 63:425 (1991), onthe basal cells within the epithelium.

B. Immunofluorescence with Anti-Ligand Antibodies

Additional sections of the human normal skin and granulation tissuesprepared above were also examined for the presence of the ligands forthe β₃ and β₁ integrins, von Willebrand factor and laminin,respectively. Von Willebrand factor localized to the blood vessels innormal skin (FIG. 2A) and granulation tissue (FIG. 2B), whereas lamininlocalized to all blood vessels as well as the epithelial basementmembrane in both tissue preparations (FIGS. 2C and 2D).

C. Distribution Anti-α_(v)β₃ Antibodies on Cancer Tissue

In addition to the above analyses, biopsies of cancer tissue from humanpatients were also examined for the presence and distribution ofα_(v)β₃. The tissues were prepared as described Example 1A with theexception that they were stained with monoclonal antibody LM609 preparedin Example 2 that is specific for the integrin receptor complex,α_(v)β₃. In addition, tumors were also prepared for microscopichistological analysis by fixing representative examples of tumors inBulins Fixative for 8 hours and serial sections cut and H&E stained.

The results of immunoperoxidase staining of bladder, colon breast andlung cancer tissues are shown in FIGS. 3A-3D, respectively. α_(v)β₃ isabundantly expressed only on the blood vessels present in the fourcancer biopsies analyzed and not on any other cells present in thetissue.

The results described herein thus show that the α_(v)β₃ integrinreceptor is selectively expressed in specific tissue types, namelygranulated, metastatic tissues and other tissues in which angiogenesisis occurring and not normal tissue where the formation of new bloodvessels has stopped. These tissues therefore provide an ideal target fortherapeutic aspects of this invention.

4. Identification of α_(v)β₃-Specific Synthetic Peptides Detected by aLigand-Receptor Binding Assay

The synthetic peptides prepared in Example 1 were screened by measuringtheir ability to antagonize α_(v)β₃ and α_(IIb)β₃ receptor bindingactivity in purified ligand-receptor binding assays. The method forthese binding studies has been described by Barbas et al., Proc. Natl.Acad. Sci., USA. 90:10003-10007 (1993) and Smith et al., J. Biol. Chem.,265:11008-11013 (1990), the disclosures of which are hereby incorporatedby reference.

Briefly, selected purified integrins were separately immobilized inTitertek microtiter wells at a coating concentration of 50 nanograms(ng) per well. The purification of the receptors used in theligand-receptor binding assays are well known in the art and are readilyobtainable with methods familiar to one of ordinary skill in the art.After incubation for 18 hours at 4 C, nonspecific binding sites on theplate were blocked with 10 milligrams/milliliter (mg/ml) of bovine serumalbumin (BSA) in Tris-buffered saline. For inhibition studies, variousconcentrations of selected peptides from Table 1 were tested for theability to block the binding of ¹²⁵I-vitronectin or ¹²⁵I-fibrinogen tothe integrin receptors, α_(v)β₃ and α_(IIb)β₃. Although these ligandsexhibit optimal binding for a particular integrin, vitronectin forα_(v)β₃ and fibrinogen for α_(IIb)β₃, inhibition of binding studiesusing peptides to block the binding of fibrinogen to either receptorallowed for the accurate determination of the amount in micromoles (uM)of peptide necessary to half-maximally inhibit the binding of receptorto ligand. Radiolabeled ligands were used at concentrations of 1 nM andbinding was challenged separately with unlabeled synthetic peptides.

Following a three hour incubation, free ligand was removed by washingand bound ligand was detected by gamma counting. The data from theassays where mn selected cyclic peptides listed in Table 1 were used toinhibit the binding of receptors and radiolabeled fibrinogen toseparately immobilized α_(v)β₃ and α_(IIb)β₃ receptors were highlyreproducible with the error between data points typically below 11%. TheIC₅₀ data in micromoles (IC₅₀ uM) are expressed as the average ofduplicate data points ± the standard deviation as shown in Table 2.

TABLE 2 Peptide No. α_(v)β₃ (IC₅₀ uM) α_(IIb)β₃ (IC₅₀ uM) 62181 1.96 ±0.62 14.95 ± 7.84  62184  0.05 ± 0.001 0.525 ± 0.10  62185 0.885 ± 0.16   100 ± 0.001 62187  0.05 ± 0.001  0.26 ± 0.056 62186 57.45 ± 7.84   100 ± 0.001 62175 1.05 ± 0.07 0.63 ± 0.18 62179 0.395 ± .21  0.055 ±0.007

Thus, the RGD-containing or RGD-derivatized cyclized peptides 62181,62184, 62185 and 62187, each having one D-amino acid residue, exhibitedpreferential inhibition of fibrinogen binding to the α_(v)β₃ receptor asmeasured by the lower concentration of peptide required for half-maximalinhibition as compared to that for the α_(IIb)β₃ receptor. In contrast,the other RGD-containing or RGD-derivatized cyclic peptides, 62186,62175 and 62179, were not as effective in blocking fibrinogen binding toα_(v)β₃, with the latter two peptides exhibiting preferential inhibitionof fibrinogen binding to α_(IIb)β₃ as compared to α_(v)β₃.

Similar inhibition of binding assays were performed with linearizedpeptides having or lacking an RGD motif, the sequences of which werederived from the α_(v) receptor subunit, α_(IIb) receptor subunit orvitronectin ligand amino acid residue sequences. The sequences of thelinear peptides, 62880 (VN-derived amino acid residues 35-49), 62411(α_(v)-derived amino acid residues 676-687); 62503 (α_(v)-derived aminoacid residues 655-667) and 62502 (α_(IIb)-derived amino acid residues296-306), are listed in Table 1. Each of these peptides were used inseparate assays to inhibit the binding of either vitronectin (VN) orfibrinogen (FG) to either α_(IIb)β₃ or α_(v)β₃. The IC₅₀ data inmicromoles (IC₅₀ uM) of an individual assay for each experiment is shownin Table 3.

TABLE 3 α_(IIb)β₃ α_(v)β₃ Peptide No. FG VN FG VN 62880 4.2 0.98 <0.10.5 62411 >100 >100 >100 >100 62503 >100 >100 >100 >100 62502 90 5 >100>100

The results of inhibition of ligand binding assays to selected integrinreceptors with linearized peptides show that only peptide 62880 waseffective at inhibiting the half-maximal binding of either FG or VN toα_(v)β₃ as measured by the lower concentration of peptide required forhalf-maximal inhibition as compared to α_(IIb)β₃ receptor. None of theother linearized peptides were effective at blocking ligand binding toα_(v)β₃ although peptide 62502 was effective at blocking VN binding toα_(IIb)β₃.

Thus, the ligand-receptor assay described herein can be used to screenfor both circular or linearized synthetic peptides that exhibitselective specificity for a particular integrin receptor, specificallyα_(v)β₃, as used as vitronectin receptor (α_(v)β₃) antagonists inpracticing this invention.

5. Characterization of the Untreated Chick Chorioallantoic Membrane(CAM)

A. Preparation of the CAM

Angiogenesis can be induced on the chick chorioallantoic membrane (CAM)after normal embryonic angiogenesis has resulted in the formation ofmature blood vessels. Angiogenesis has been shown to be induced inresponse to specific cytokines or tumor fragments as described byLeibovich et al., Nature, 329:630 (1987) and Ausprunk et al., Am. J.Pathol., 79:597 (1975). CAMs were prepared from chick embryos forsubsequent induction of angiogenesis and inhibition thereof as describedin Examples 6 and 7, respectively. Ten day old chick embryos wereobtained from McIntyre Poultry (Lakeside, Calif.) and incubated at 99.5degrees Fahrenheit with 60% humidity. A small hole was made through theshell at the end of the egg directly over the air sac with the use of asmall crafts drill (Dremel, Division of Emerson Electric Co. RacineWis.). A second hole was drilled on the broad side of the egg in aregion devoid of embryonic blood vessels determined previously bycandling the egg. Negative pressure was applied to the original hole,which resulted in the CAM (chorioallantoic membrane) pulling away fromthe shell membrane and creating a false air sac over the CAM. A 1.0centimeter (cm)×1.0 cm square window was cut through the shell over thedropped CAM with the use of a small model grinding wheel (Dremel). Thesmall window allowed direct access to the underlying CAM.

The resultant CAM preparation was then either used at 6 days ofembryogenesis, a stage marked by active neovascularization, withoutadditional treatment to the CAM reflecting the model used for evaluatingeffects on embryonic neovascularization or used at 10 days ofembryogenesis where angiogenesis has subsided. The latter preparationwas thus used in this invention for inducing renewed angiogenesis inresponse to cytokine treatment or tumor contact as described in Example6.

B. Histology of the CAM

To analyze the microscopic structure of the chick embryo CAMs and/orhuman tumors that were resected from the chick embryos as described inExample 8, the CAMs and tumors were prepared for frozen sectioning asdescribed in Example 3A. Six micron thick sections were cut from thefrozen blocks on a cryostat microtome for immunofluorescence analysis.

FIG. 4 shows a typical photomicrograph of an area devoid of bloodvessels in an untreated 10 day old CAM. As angiogenesis in the CAMsystem is subsiding by this stage of embryogenesis, the system is usefulin this invention for stimulating the production of new vasculature fromexisting vessels from adjacent areas into areas of the CAM currentlylacking any vessels.

C. Integrin Profiles in the CAM Detected by Immunofluorescence

To view the tissue distribution of integrin receptors present in CAMtissues, 6 micron (um) frozen sections of both tumor tissue and chickembryo CAM tissues were fixed in acetone for 30 seconds and stained byimmunofluorescence with 10 micrograms/milliliter (ug/ml) mAb CSAT, amonoclonal antibody specific for the β₁ integrin subunit as described byBuck et al., J. Cell Biol., 107:2351 (1988) and thus used for controls,or LM609 as prepared in Example 2. Primary staining was followed bystaining with a 1:250 dilution of goat anti-mouse rhodamine labeledsecondary antibody (Tango) to allow for the detection of the primaryimmunoreaction product. The sections were then analyzed with a Zeissimmunofluorescence compound microscope.

The results of the immunofluorescence analysis show that the matureblood vessels present in an untreated 10 day chick embryo expressed theintegrin β₁ subunit (FIG. 5A). In contrast, in a serial section of thetissue shown in FIG. 5A, no immunoreactivity with LM609 was revealed(FIG. 5B). Thus, the integrin α_(v)β₃ detected by the LM609 antibody wasnot actively being expressed by the mature blood vessels present in a 10day old untreated chick embryo. As shown in the CAM model and in thefollowing Examples, while the blood vessels are undergoing new growth innormal embryogenesis or induced by either cytokines or tumors, the bloodvessels are expressing α_(v)β₃. However, following activeneovascularization, once the vessels have stopped developing, theexpression of α_(v)β₃ diminishes to levels not detectable byimmunofluorescence analysis. This regulation of α_(v)β₃ expression inblood vessels undergoing angiogenesis as contrasted to the lack ofexpression in mature vessels provides for the unique ability of thisinvention to control and inhibit angiogenesis as shown in the followingExamples as modeled using the CAM angiogenesis assay system.

6. CAM Angiogenesis Assay

A. Angiogenesis Induced by Growth Factors

Angiogenesis has been shown to be induced by cytokines or growth factorsas referenced in Example 5A. In the experiments described herein,angiogenesis in the CAM preparation described in Example 5 was inducedby growth factors that were topically onto the CAM blood vessels asdescribed herein.

Angiogenesis was induced by placing a 5 millimeter (mm)×5 mm Whatmanfilter disk (Whatman Filter paper No.1.) saturated with Hanks BalancedSalt Solution (HBSS) or HBSS containing 150 nanograms/milliliter (ng/ml)recombinant basic fibroblast growth factor (βFGF) (Genzyme, Cambridge,Mass.) on the CAM of a 10-day chick embryo in a region devoid of bloodvessels and the windows were latter sealed with tape. In other assays,125 ng/ml βFGF was also effective at inducing blood vessel growth.Angiogenesis was monitored by photomicroscopy after 72 hours. CAMs weresnap frozen, and 6 um cryostat sections were fixed with acetone andstained by immunofluorescence as described in Example 5C with 10 ug/mlof either anti-β₁ monoclonal antibody CSAT or LM609.

The immunofluorescence photomicrograph in FIG. 5C shows enhancedexpression of α_(v)β₃ during βFGF-induced angiogenesis on the chick CAMin contrast with the absence of α_(v)β₃ expression in an untreated chickCAM as shown in FIG. 5B. α_(v)β₃ was readily detectable on many (75% to80%) of the vessels on the βFGF-treated CAMs. In addition, theexpression of integrin β₁ did not change from that seen in an untreatedCAM as β₁ was also readily detectable on stimulated blood vessels.

The relative expression of α_(v)β₃ and β₁ integrins were then quantifiedduring βFGF-induced angiogenesis by laser confocal image analysis of theCAM cryostat sections. The stained sections were then analyzed with aZeiss laser confocal microscope. Twenty-five vessels stained with LM609and 15 stained with CSAT (average size ˜1200 sq mm², range 350 to 3,500mm²) were selected from random fields and the average rhodaminefluorescence for each vessel per unit area was measured in arbitraryunits by laser confocal image analysis. Data are expressed as the meanfluorescence intensity in arbitrary units of vessels ± standard error(SE).

The results plotted in FIG. 6 show that staining of α_(v)β₃ wassignificantly enhanced (four times higher) on CAMs treated with βFGF asdetermined by the Wilcoxon Rank Sum Test (P<0.0001) whereas β₁ stainingwas not significantly different with βFGF treatment.

The CAM assay was further used to examine the effect of another potentangiogenesis inducer, tumor necrosis factor-alpha (TNFα), on theexpression of β₁ and β₃ integrins. Filter disks impregnated with eitherβFGF or TNFα and placed on CAMs from 10 day embryos were found topromote local angiogenesis after 72 hours.

The results are shown in the photomicrographs of CAMs either untreated(FIG. 7A), treated with βFGF (FIG. 7B) or treated with TNFα (FIG. 7C).Blood vessels are readily apparent in both the βFGF and TNFα treatedpreparations but are not present in the untreated CAM. Thus, the topicalapplication of a growth factor/cytokine resulted in the induction ofangiogenesis from mature vessels in an adjacent area into the areaoriginally devoid of blood vessels. In view of the βFGF-induced bloodvessels and concomitant expression of α_(v)β₃ as shown in FIG. 5C,treatment of TNFα results in comparable activities.

These findings indicate that in both human and chick, blood vesselsinvolved in angiogenesis show enhanced expression of α_(v)β₃. Consistentwith this, expression of α_(v)β₃ on cultured endothelial cells can beinduced by various cytokines in vitro as described by Janat et al., J.Cell Physiol., 151:588 (1992); Enenstein et al., Exp. Cell Res., 203:499(1992) and Swerlick et al., J. Invest. Derm., 99:715 (1993).

The effect on growth-factor induced angiogenesis by antibody and peptideinhibitors is presented in Examples 7A and 7B.

B. Embryonic Angiogenesis

The CAM preparation for evaluating the effect of angiogenesis inhibitorson the natural formation of embryonic neovasculature was the 6 dayembryonic chick embryo as previously described. At this stage indevelopment, the blood vessels are undergoing de novo growth and thusprovides a useful system for determining if α_(v)β₃ participates inembryonic angiogenesis. The CAM system was prepared as described abovewith the exception that the assay was performed at embryonic day 6rather than at day 10. The effect on embryonic angiogenesis by treatmentwith antibodies and peptides of this invention are presented in Example7C.

C. Angiogenesis Induced by Tumors

To investigate the role of α_(v)β₃ in tumor-induced angiogenesis,α_(v)β₃-negative human M21-L melanoma fragments were used in the CAMassay that were previously grown and isolated from the CAM of a 17-daychick embryo as described by Brooks et al., J. Cell Biol., 122:1351(1993) and as described herein. These fragments induced extensiveneovascularization in the presence of buffer alone.

Angiogenesis was induced in the CAM assay system by direct apposition ofa tumor fragment on the CAM. Preparation of the chick embryo CAM wasidentical to the procedure described above. Instead of a filter paperdisks a 50 milligram (mg) to 55 mg in weight fragment of either humanmelanoma tumor M21L or human lung carcinoma tumor UCLAP-3, both of whichare α_(v)β₃ negative tumors, was placed on the CAM in an area originallydevoid of blood vessels.

The M21L human melanoma cell line or the UCLAP-3 human lung carcinomacell line, both α_(v)β₃ negative, were used to grow the solid humantumors on the CAMs of chick embryos. A single cell suspension of 5×10⁶M21L or UCLAP-3 cells were first applied to the CAMs in a total volumeof 30 microliters (ul) of sterile HBSS. The windows were sealed withtape and the embryos were incubated for 7 days to allow growth of humantumor lesions. At the end of 7 days, now a 17-day embryo, the tumorswere resected from the CAMs and trimmed free of surrounding CAM tissue.The tumors were sliced into 50 mg to 55 mg tumor fragments. The tumorfragments were placed on a new set of 10 day chick embryo CAMs asdescribed in Example 6A in an area devoid of blood vessels.

These CAM tumor preparations were then subsequently treated as describedin Examples 7D and 7E for measuring the effects of antibodies andpeptides on tumor-induced angiogenesis.

7. Inhibition of Angiogenesis as Measured in the CAM Assay

A. Inhibition of Growth Factor-Induced Angiogenesis by TopicalApplication of Inhibitors

1) Treatment with Monoclonal Antibodies

To determine whether α_(v)β₃ plays an active role in angiogenesis,filter disks saturated with βFGF or TNFα were placed on CAMs then themonoclonal antibodies (also referred to as mAb), LM609 (specific forα_(v)β₃), CSAT (specific for β₁) or P3G2 (specific for α_(v)β₃) wereadded to the preparation.

Angiogenesis was induced on CAMs from 10 day chick embryos by filterdisks saturated with βFGF. Disks were then treated with 50 ml HBSScontaining 25 mg of mAb in a total volume of 25 ul of sterile HBSS at 0,24, and 48 hours. At 72 hours, CAMs were harvested and placed in a 35 mmpetri dish and washed once with 1 ml of phosphate buffered saline. Thebottom side of the filter paper and CAM tissue was then analyzed underan Olympus stereo microscope, with two observers in a double-blindfashion. Angiogenesis inhibition was considered significant when CAMsexhibited >50% reduction in blood vessel infiltration of the CAMdirectly under the disk. Experiments were repeated four times perantibody, with 6 to 7 embryos per condition.

The results of the effects of mAb treatment on βFGF-induced angiogenesisis shown in FIGS. 8A-8B. An untreated CAM preparation devoid of bloodvessels is shown in FIG. 8A to provide a comparison with the βFGF-bloodvessel induction shown in FIG. 8B and effects thereon by the mAbs inFIGS. 8C-8E. About 75% of these CAMs treated with mAb LM609exhibited >50% inhibition of angiogenesis as shown in FIG. 8E, and manyof these appeared devoid of vessel infiltration. In contrast, the buffercontrol (FIG. 8A) and disks treated with mAbs CSAT (FIG. 8C) and P3G2(FIG. 8D) consistently showed extensive vascularization.

Identical results were obtained when angiogenesis was induced with TNFα.To examine the effects of these same antibodies on preexisting matureblood vessels present from normal vessel development adjacent to theareas devoid of vessels, filter disks saturated with mAbs were placed onvascularized regions of CAMs from 10 day embryos that did not receivetopical application of cytokine. None of the three mabs affectedpreexisting vessels, as assessed by visualization under a stereomicroscope. Thus, mAb LM609 selectively inhibited only new blood vesselgrowth and did not effect mature blood vessels present in adjacentareas. This same effect was seen with the application of syntheticpeptides either applied topically or intravenously as described inExamples 7A2) and 7E2), respectively.

2) Treatment with Synthetic Peptides

CAM assays were also performed with the synthetic peptides of thisinvention to determine the effect of cyclic and linearized peptides ongrowth factor induced angiogenesis. The peptides were prepared asdescribed in Example 1 and 80 ug of peptide was presented in a totalvolume of 25 ul of sterile HBSS. The peptide solution was applied to theCAM preparation immediately and then again at 24 and 48 hrs. At 72 hoursthe filter paper and surrounding CAM tissue was dissected and viewed asdescribed above.

Results from this assay revealed were similar to those shown in FIGS.9A-9C as described in Example 7E2) where synthetic peptides wereintravenously injected into tumor induced blood vessels. Here, with thecontrol peptide, 62186, the βFGF-induced blood vessels remainedundisturbed as shown in FIG. 9A. In contrast when the cyclic RGDpeptide, 62814, was applied to the filter, the formation of bloodvessels was inhibited leaving the area devoid of new vasculature. Thiseffect was similar in appearance to that shown in FIG. 9B as describedin Example 7E2) below. In addition, also as shown in FIG. 9C forintravenously injected peptides, in areas in which mature blood vesselswere present yet distant from the placement of the growth-factorsaturated filter, no effect was seen with the topical treatment ofsynthetic peptides on these outlying vessels. The inhibitory activity ofthe peptides on angiogenesis thus is limited to the areas ofangiogenesis induced by growth factors and does not effect adjacentpreexisting mature vessels or result in any deleterious cytotoxicity tothe surrounding area.

Similar assays are performed with the other synthetic peptides preparedin Example 1 and listed in Table 1.

B. Inhibition of Growth Factor-Induced Angiogenesis by IntravenousApplication of Inhibitors

1) Treatment with Monoclonal Antibodies

The effect on growth factor-induced angiogenesis with monoclonalantibodies intravenously injected into the CAM preparation was alsoevaluated for use in this invention.

The preparation of the chick embryo CAMs for intravenous injections wereessentially as described in Example 7A with some modifications. Duringthe candling procedures prominent blood vessels were selected and markswere made on the egg shell to indicate their positions. The holes weredrilled in the shell and the CAMs were dropped and βFGF saturated filterpapers were placed on the CAMs as described above. The windows weresealed with sterile tape and the embryos were replaced in the incubator.Twenty four hours later, a second small window was carefully cut on thelateral side of the egg shell directly over prominent blood vesselsselected previously. The outer egg shell was carefully removed leavingthe embryonic membranes intact. The shell membrane was made transparentwith a small drop of mineral oil (Perkin-Elmer Corp, Norwalk, Conn.)which allowed the blood vessels to be visualized easily. Purifiedsterile MAbs, or synthetic peptides, the latter of which are describedbelow, were inoculated directly into the blood vessels once with a 30gauge needle at a dose of 200 ug of IgG per embryo in a total volume of100 ul of sterile PBS. The windows were sealed with tape and the embryoswere allowed to incubate until 72 hours. The filter disks andsurrounding CAM tissues were analyzed as described before.

To determine the localization of LM609 mAb in CAM tissues or in tumortissues, as shown herein and in the following Examples, that werepreviously inoculated intravenously with LM609, the fixed sections wereblocked with 2.5% BSA in HBSS for 1 hour at room temperature followed bystaining with a 1:250 dilution of goat anti-mouse rodamine labeledsecondary antibody (Tango). The sections were then analyzed with a Zeissimmunofluorescence compound microscope.

The results of intravenous antibody treatment to mil the βFGF inducedblood vessel CAM preparation are shown in FIGS. 10A-10C. In FIG. 10A,angiogenesis induced as a result of βFGF treatment is shown. No changeto the presence of βFGF induced vasculature was seen with intravenousexposure to mAb P3G2, an anti-α_(v)β₅ antibody, as shown in FIG. 10B. Incontrast, treatment of the βFGF induced angiogenesis CAM preparationwith LM609, an anti-α_(v)β₃ antibody, resulted in the completeinhibition of growth of new vessels into the filter area as shown inFIG. 10C. The inhibitory effect on angiogenesis is thus resulting fromthe inhibition of α_(v)β₃ receptor activity by the LM609anti-α_(v)β₃-specific antibody. Since the blocking of the α_(v)β₅ doesnot inhibit the formation of neovasculature into the CAMs filter site,α_(v)β₅, thus is not essential as compared to α_(v)β₃ for growth of newvessels.

2) Treatment with Synthetic Peptides

The synthetic peptides prepared in Example 1 are separatelyintravenously injected into the growth factor induced blood vessels inthe CAM preparation as described above. The effect of the peptides onthe viability of the vessels is similarly assessed.

C. Inhibition of Embryonic Angiogenesis by Topical Application

1) Treatment with Monoclonal Antibodies

To determine whether α_(v)β₃ participates in embryonic angiogenesis, theeffect of LM609 on de novo growth of blood vessels on CAMs was examinedin 6 day embryos, a stage marked by active neovascularization asdescribed in Example 5A. The CAM assay was prepared as described inExample 6C with the subsequent topical application of disks saturatedwith mabs placed on CAMs of 6 day old embryos in the absence ofcytokines. After 3 days, CAMS were resected and photographed. Eachexperiment included 6 embryos per group and was repeated 2 times.

Antibody LM609 (FIG. 1C), but not CSAT (FIG. 11A) or P3G2 (FIG. 11B),prevented vascular growth under these conditions; this indicates thatα_(v)β₃ plays a substantial role in embryonic neovascularization thatwas independent of added growth factors for induction of angiogenesis.

2) Treatment with Synthetic Peptides

The synthetic peptides prepared in Example 1 are separately added to theembryonic CAM preparation prepared above and as described in Example5A2) by either topical application to the CAM or intravenous applicationto blood vessels. The effect of the peptides on the viability of thevessels is similarly assessed.

D. Inhibition of Tumor-Induced Angiogenesis by Topical Application

1) Treatment with Monoclonal Antibodies

In addition to the angiogenesis assays described above where the effectsof anti-α_(v)β₃ antagonists, LM609 and peptides 62181, 62184, 62185,62187 and 62880, on embryonic angiogenesis were evaluated, the role ofα_(v)β₃ in tumor-induced angiogenesis was also investigated. As aninducer, α_(v)β₃-negative human M21-L melanoma fragments previouslygrown and isolated from the CAM of a 17-day chick embryo were used. Thefragments were prepared as described in Example 6C.

As described above in Example 7A1, mAbs were separately topicallyapplied to the tumor fragments at a concentration of 25 ug in 25 ul ofHBSS and the windows were then sealed with tape. The mAbs were addedagain in the same fashion at 24 hours and 48 hours. At 72 hours, thetumors and surrounding CAM tissues were analyzed as described above inExample 7A1).

As described in Example 6C, tumors were initially derived bytransplanting cultured M21-L cells, which do not to express integrinα_(v)β₃ as described by Felding-Habermann et al., J. Clin. Invest.,89:2018 (1992) onto the CAMs of 10-day old chick embryos. Theseα_(v)β₃-negative fragments induced extensive neovascularization in thepresence of buffer alone, or mAbs CSAT (anti-β₁) or P3G2 (anti-α_(v)β₅).In contrast, mAb LM609 (anti-α_(v)β₃) abolished the infiltration of mostvessels into the tumor mass and surrounding CAM.

In order to quantitate the effect of the mAbs on the tumor-inducedangiogenesis, blood vessels entering the tumor within the focal plane ofthe CAM were counted under a stereo microscope by two observers in adouble-blind fashion. Each data bar presented in FIG. 12 represents themean number of vessels ± SE from 12 CAMs in each group representingduplicate experiments.

This quantitative analysis revealed a three-fold reduction in the numberof vessels entering tumors treated with Mab LM609 compared to tumorstreated with buffer or the other mAbs, P3G2 or CSAT (P<0.0001) asdetermined by Wilcoxon Rank Sum Test. The fact that M21-L tumors do notexpress α_(v)β₃ indicates that mAb LM609 inhibits angiogenesis bydirectly affecting blood vessels rather than the tumor cells. Theseresults correspond with the histological distribution of α_(v)β₃ incancer tissue biopsies shown in FIG. 3A-3D where the distribution ofα_(v)β₃ was limited to the blood vessels in the tumor and not to thetumor cells themselves.

2) Treatment with Synthetic Peptides

The synthetic peptides prepared in Example 1 are topically applied tothe tumor-induced angiogenic CAM assay system as described above. Theeffect of the peptides on the viability of the vessels is similarlyassessed.

E. Inhibition of Tumor-Induced Angiogenesis by Intravenous Application

1) Treatment with Monoclonal Antibodies

Tumor-induced blood vessels prepared as described in Example 7D1) werealso treated with mAbs applied by intravenous injection. Tumors wereplaced on the CAMs as described in Example 7D1) and the windows sealedwith tape and 24 hours latter, 200 ug of purified mAbs were inoculatedonce intravenously in chick embryo blood vessels as describedpreviously. The chick embryos were then allowed to incubate for 7 days.The extent of angiogenesis was then observed as described in above. Asdescribed in Example 8 below, after this time period, the tumors wereresected and analyzed by their weight to determine the effect ofantibody exposure on tumor growth or suppression.

2) Treatment with Synthetic Peptides

The effects of peptide exposure to tumor-induced vasculature in the CAMassay system was also assessed. The tumor-CAM preparation was used asdescribed above with the exception that instead of intravenous injectionof a mAb, synthetic peptides prepared as described in Example 1 andExample 7A2) were separately intravenously injected into visible bloodvessels.

The results of CAM assays with the cyclic peptide, 66203 containing theHCl salt, and control peptide, 62186, are shown in FIGS. 9A-9C. In FIG.9A, the treatment with the control peptide did not effect the abundantlarge blood vessels that were induced by the tumor treatment to growinto an area originally devoid of blood vessels of the CAM. In contrastwhen the cyclic RGD peptide, 66203, an antagonist to α_(v)β₃, wasapplied to the filter, the formation of blood vessels was inhibitedleaving the area devoid of new vasculature as shown in FIG. 9B. Theinhibitory effect of the RGD-containing peptide was specific andlocalized as evidenced by an absence of any deleterious effects tovessels located adjacent to the tumor placement. Thus, in FIG. 9C, wheninhibitory peptides are intravenously injected into the CAM assaysystem, no effect was seen on the preexisting mature vessels present inthe CAM in areas adjacent yet distant from the placement of the tumor.The preexisting vessels in this location were not affected by theinhibitory peptide that flowed within those vessels although thegeneration of new vessels from these preexisting vessels into the tumormass was inhibited. Thus, synthetic peptides including 66203 and 62184,previously shown in ligand-receptor assays in Example 4 to beantagonists of α_(v)β₃, have now been demonstrated to inhibitangiogenesis that is limited to vessels undergoing development and notto mature preexisting vessels. In addition, the intravenous infusion ofpeptides does not result in any deleterious cytotoxicity to thesurrounding area as evidence by the intact vasculature in FIG. 9C.

Similar assays are performed with the other synthetic peptides preparedin Example 1 and listed in Table 1.

8. Inhibition of Tumor Tissue Growth with α_(v)β₃ Antagonists

As described in Example 7D1), in addition to visually assessing theeffect of anti-α_(v)β₃ antagonists on growth factor or tumor inducedangiogenesis, the effect of the antagonists was also assessed bymeasuring any changes to the tumor mass following exposure. For thisanalysis, the tumor-induced angiogenesis CAM assay system was preparedas described in Example 6C and 7D. At the end of the 7 day incubationperiod, the resulting tumors were resected from the CAMs and trimmedfree of any residual CAM tissue, washed with 1 ml of phosphate buffersaline and wet weights were determined for each tumor. In addition,preparation of the tumor for microscopic histological analysis includedfixing representative examples of tumors in Bulins Fixative for 8 hoursand serial sections cut and H&E stained.

A. Topical Application

The results of typical human melanoma tumor (M21L) weights resultingfrom topical application of control buffer (HBSS), P3G2 (anti-α_(v)β₅)or LM609 (anti-α_(v)β₃) are listed in Table 4. A number of embryos wereevaluated for each treatment with the average tumor weight in milligrams(mg) from each being calculated along with the SE of the mean as shownat the bottom of the table.

TABLE 4 Embryo No. mAb Treatment Tumor Weight (mg) 1 HBSS 108 2 152 3216 4 270 5 109 6 174 1 P3G2 134 2 144 3 408 4 157 5 198 6 102 7 124 8 99 1 LM609  24 2 135 3  17 4  27 5  35 6  68 7  48 8  59 mAb TreatmentAverage Tumor Weight HBSS control 172 ± 26 P3G2 171 ± 36 LM609  52 ± 13

Exposure of a α_(v)β₃-negative human melanoma tumor mass in the CAMassay system to LM609 caused the decrease of the untreated average tumorweight of 172 mg ±26 to 52 mg ±13. The P3G2 antibody had no effect onthe tumor mass. Thus, the blocking of the α_(v)β₃ receptor by thetopical application of α_(v)β₃-specific LM609 antibody resulted in aregression of tumor mass along with an inhibition of angiogenesis asshown in the preceding Examples. The measured diameter of the tumor massresulting from exposure to P3G2 was approximately 8 millimeters to 1centimeter on average. In contrast, the LM609-treated tumors were onaverage 2 to 3 millimeters in diameter.

Frozen sections of these tumors revealed an intact tumorcytoarchitecture for the tumor exposed to P3G2 in contrast to a lack ororganized cellular structure in the tumor exposed to LM609. α_(v)β₃receptor activity is therefore essential for an α_(v)β₃ negative tumorto maintain its mass nourished by development of α_(v)β₃-expressingneovasculature. The blocking of α_(v)β₃ with the α_(v)β₃ antagonists ofthis invention results in the inhibition of angiogenesis into the tumorultimately resulting in the dimunition of tumor mass.

B. Intravenous Application

The results of typical carcinoma tumor (UCLAP-3) weights resulting fromintravenous application of control buffer (PBS, phosphate bufferedsaline), CSAT (anti-β₁) or LM609 (anti-α_(v)β₃) are listed in Table 5. Anumber of embryos were evaluated for each treatment with the averagetumor weight from each being calculated along with the SE of the mean asshown at the bottom of the table.

TABLE 5 Embryo No. mAb Treatment Tumor Weight 1 PBS 101 2  80 3  67 4 90 1 CSAT 151 2  92 3 168 4  61 5  70 1 LM609  16 2  54 3  30 4  20 5 37 6  39 7  12 mAb Treatment Average Tumor Weight PBS control 85 ± 7 CSAT 108 ± 22  LM609 30 ± 6 

Exposure of α_(v)β₃-negative human carcinoma tumor mass in the CAM assaysystem to LM609 caused the decrease of the untreated average tumorweight of 85 mg ±7 to 30 mg ±6. The CSAT antibody did not significantlyeffect the weight of the tumor mass. Thus, the blocking of the α_(v)β₃receptor by the intravenous application of α_(v)β₃-specific LM609antibody resulted in a regression of a carcinoma as it did for themelanoma tumor mass above along with an inhibition of angiogenesis asshown in the preceding Examples. In addition, human melanoma tumorgrowth was similarly inhibited by intravenous injection of LM609.

Thus, the aforementioned Examples demonstrate that integrin α_(v)β₃plays a key role in angiogenesis induced by a variety of stimuli and assuch α_(v)β₃ is a valuable therapeutic target with the α_(v)β₃antagonists of this invention for diseases characterized byneovascularization.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the cell line deposited,since the deposited embodiment is intended as a single illustration ofone aspect of the invention and any cell line that is functionallyequivalent is within the scope of this invention. The deposit ofmaterial does not constitute an admission that the written descriptionherein contained is inadequate to enable the practice of any aspect ofthe invention, including the best mode thereof, nor is it to beconstrued as limiting the scope of the claims to the specificillustration that it represents. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims.

1. A method of inducing tumor tissue regression in a patient comprising administering to said patient a therapeutically effective amount of an antibody immunospecific for α_(v)β₃.
 2. The method of claim 1 wherein said antibody is a monoclonal antibody.
 3. The method of claim 1 or 2 wherein said tissue is a solid tumor tissue.
 4. The method of claim 1 or 2 wherein said tumor tissue is bladder.
 5. The method of claim 1 or 2 wherein said tumor tissue is breast.
 6. The method of claim 1 or 2 wherein said tumor tissue is colon.
 7. The method of claim 1 or 2 wherein said tumor tissue is lung.
 8. The method of claim 1 or 2 wherein said tumor tissue is skin.
 9. The method of claim 1 or 2 wherein said tumor tissue is a carcinoma.
 10. The method of claim 1 or 2 wherein said tumor tissue is a melanoma.
 11. The method of claim 1 or 2 wherein said administering comprises intravenous administration.
 12. The method of claim 1 or 2 wherein said administering comprises oral administration.
 13. The method of claim 1 or 2 wherein said administering comprises transdermal administration.
 14. The method of claim 1 or 2 wherein said administering comprises intramuscular administration.
 15. The method of claim 1 or 2 wherein said administering comprises subcutaneous administration.
 16. The method of claim 1 or 2 wherein said administering comprises intracavity administration.
 17. The method of claim 1 or 2 wherein said administering comprises peristaltic administration.
 18. The method of claim 1 or 2 wherein said administering comprises parenteral administration.
 19. The method of claim 1 or 2 wherein said administering comprises systemic administration.
 20. The method of claim 1 or 2 wherein said administering comprises administration by gradual infusion.
 21. The method of claim 1 or 2 wherein said administering comprises one or more dose administrations daily, for one or several days.
 22. The method of claim 1 or 2 wherein said administering comprises a single dose.
 23. The method of claim 1 or 2 wherein said amount is from about 0.1 mg/kg to about 300 mg/kg patient body weight.
 24. The method of claim 1 or 2 wherein said amount is from about 0.2 mg/kg to about 200 mg/kg patient body weight.
 25. The method of claim 1 or 2 wherein said amount is from about 0.5 mg/kg to about 20 mg/kg patient body weight.
 26. The method of claim 1 or 2 wherein said administering is conducted in conjunction with chemotherapy.
 27. The method of claim 1 or 2 wherein the patient is human.
 28. The method of claim 1 or 2 wherein the patient is a patient with angiofibromas.
 29. The method of claim 1 or 2 wherein the patient is a patient with retrolental fibroplasia.
 30. The method of claim 1 or 2 wherein the patient is a patient with hemangiomas.
 31. The method of claim 1 or 2 wherein the patient is a patient with Karposi sarcoma.
 32. The method of claim 1 or 2 wherein said antibody preferentially inhibits fibrinogen binding to α_(v)β₃ compared to fibrinogen binding to α_(IIb)β₃.
 33. The method of claim 1 or 2 wherein said antibody specifically binds α_(v)β₃ complex.
 34. The method of claim 1 or 2 wherein said antibody is an antibody fragment.
 35. The method of claim 34 wherein said antibody is a Fab fragment.
 36. The method of claim 34 wherein said antibody is a Fab′ fragment.
 37. The method of claim 34 wherein said antibody is a F(ab′)₂ fragment.
 38. The method of claim 34 wherein said antibody is a F(v) fragment.
 39. The method of claim 2 wherein said monoclonal antibody has the binding specificity of the monoclonal antibody LM609 having ATCC accession number HB
 9537. 40. The method of claim 2 wherein said monoclonal antibody is humanized.
 41. The method of claim 1 or 2 wherein said tumor tissue is metastatic.
 42. The method of claim 1 or 2 wherein said antibody is administered in a composition.
 43. The method of claim 42 wherein said composition is a sterile pharmaceutical composition.
 44. The method of claim 1 or 2 wherein said administering is conducted following surgery.
 45. The method of claim 1 or 2 wherein said patient was previously treated for a first tumor.
 46. The method of claim 45 wherein said first tumor is a solid tumor.
 47. The method of claim 45 wherein said first tumor is a carcinoma.
 48. The method of claim 45 wherein said first tumor is a melanoma.
 49. The method of claim 45 wherein said patient previously underwent surgery to remove said first tumor.
 50. The method of claim 45 wherein said patient was previously treated with chemotherapy.
 51. A method of inducing solid tumor tissue regression in a human patient in need thereof, said method comprising administering to said human patient a therapeutically effective amount of a humanized monoclonal antibody immunospecific for α_(v)β₃ complex.
 52. The method of claim 51 wherein said humanized monoclonal antibody has the binding specificity of the monoclonal antibody LM609 having ATCC accession number HB9537.
 53. The method of claim 51 or 52 wherein said tumor tissue is a melanoma.
 54. The method of claim 51 or 52 wherein said tumor tissue is a carcinoma.
 55. The method of claim 51 or 52 wherein said tumor tissue is a skin carcinoma solid tissue tumor.
 56. The method of claim 51 or 52 wherein said tumor tissue is a bladder carcinoma solid tissue tumor.
 57. The method of claim 51 or 52 wherein said tumor tissue is a breast carcinoma solid tissue tumor.
 58. The method of claim 51 or 52 wherein said tumor tissue is a colon carcinoma solid tissue tumor.
 59. The method of claim 51 or 52 wherein said tumor tissue is a lung carcinoma solid tissue tumor.
 60. The method of claim 1, 2 or 51 wherein said antibody preferentially binds α_(v)β₃ over other integrins.
 61. The method of claim 1, 2 or 51 wherein said antibody does not immunoreact with a β₃ subunit.
 62. The method of claim 1, 2 or 51 wherein said antibody does not immunoreact with an α_(v) subunit.
 63. The method of claim 1, 2 or 51 wherein said antibody does not immunoreact with integrins other than α_(v)β₃. 